Errors in chromosome segregation cause birth defects and genetic Instability in tumor cells. The spindle checkpoint reduces these errors by keeping cells from starting chromosome segregation until all their chromosomes have been properly aligned on the mitotic spindle. Defects in this checkpoint are found in a large fraction of colon cancers, and are likely to play an important role in tumor initiation and progression. This application proposes genetic, cell biological, and biochemical studies of the spindle checkpoint. Their goal is to elucidate the following key steps in this regulatory pathway: how it detects misaligned chromosomes, how the detector generates a biochemical signal, how this signal inhibits the machinery that initiates chromosome segregation and cell division, and how cells eventually adapt to this signal and divide despite the presence of persistent spindle damage. The experiments are designed to take advantage of the different strengths of budding yeast, frog egg extracts, and tissue culture cells for studying the spindle checkpoint. Experiments are proposed to: 1) Determine whether the checkpoint monitors attachment of microtubules to the kinetochores or the amount of tension at the kinetochore (the microtubule-binding region of the chromosome). 2) To identify checkpoint components that act at the kinetochore to monitor its interactions with microtubules. 3) To use biochemical and genetic strategies to determine how the checkpoint generates a signal that arrests the cell division cycle, determine how this signal inhibits the proteolysis machinery that induces sister chromatid separation, and to determine how the activity of the checkpoint is regulated during the cell division cycle. 4) To determine how cells modulate the activity of the checkpoint. Recovery is defined as the reduction in the output of the checkpoint that occurs after a transient defect in the spindle has been repaired and adaptation is defined as the slow reduction in the output of the checkpoint in cells that have persistent spindle defects. 5) To identify small molecule and peptide inhibitors of the spindle checkpoint. These inhibitors will be useful as research tools in organisms that lack sophisticated genetics, will identify new components of the checkpoint, and may represent a novel class of chemotherapeutic agents.
